PPRROOJJEECCTT SSUUMMMMAARRYY This project will establish a new technology that will enable simultaneous multi-parameter phenotyping of 100s of gene knockouts. This will greatly advance annotation of the genome, and help identify genes important for numerous facets of cancer biology. There are more than 23,000 protein-coding genes in the human genome, as well as 100s of non-coding RNA genes, including microRNAs. Though there has been progress in assigning some function to many genes, we still do not know the function of many genes, and we do not know the role of most genes in driving or affecting disease. Solving this problem requires new technologies that enable high-throughput genetic screens that incorporate high content phenotyping. Over the past few years pooled genetic screens utilizing shRNAs, and now CRISPRs, have emerged as a powerful and widely used method for identifying genes involved in different aspects of cancer, including cell growth and drug resistance. Unfortunately, the current technology, which utilizes DNA as a barcode and deep- sequencing for de-convolution, has major limitations. For one, the read-out is performed on bulk cells, which means single cells cannot be analyzed with this technology. Another limitation is that DNA barcoding requires selection of cells based on single phenotypes, predominately cell fitness (do cell's survive or proliferate better). More informative phenotypes, such as upregulation or downregulation of key proteins, cannot be easily assessed with current screening technology. The objective of this project is to establish a new system of cell barcoding for vector and cell tracking. We hypothesize that combinations of linear epitopes can be used to generate protein barcodes (Pro-Codes), and Pro-Codes will enable high content single cell phenotypic analysis of 100s of gene knock-outs simultaneously. In preliminary studies we established that 10 linear epitopes can be combinatorially assembled to generate 120 different barcodes, which are easily distinguished in cells, at single cell resolution, by FACS and mass cytometry (CyTOF), with a mere 10 antibodies. We will now: (1) validate and further optimize the Pro-Code technology for simultaneous single-cell tracking, including generation of sophisticated software for Pro-Code analysis, (2) establish the Pro-Code technology for CRISPR knockout and multiparameter phenotyping, and utilize it to identify genes that influence T cell activation, and (3) expand the technology to >1,000 Pro-Codes and establish their use for double knockout screens. The outcome of this project will be a technology that enables high-content annotations of gene functions. This can help transform cancer research by accelerating the discovery of new drug targets for controlling cancer growth and immune modulation, as well as identifying mechanisms of cancer biology.